Stable lipid-comprising drug delivery complexes and methods for their production

ABSTRACT

Novel stable, concentrated, biologically active and ready-to-use lipid-comprising drug delivery complexes and methods for their production are described. The biological activity of the complexes produced are comparable to the formulations prepared according to the prior art admixture method and upon purification, the complexes produced by the method of this invention are 50 to 500 fold more concentrated than the complexes formed by admixture. The method described herein provides for the large scale production of lipid-comprising drug delivery systems useful for gene therapy and other applications.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/943,434, filed on Nov. 20, 2007, now U.S. Pat. No. 7,655,468, issuedFeb. 2, 2010, which is a continuation of U.S. patent application Ser.No. 11/014,528, filed on Dec. 15, 2004, now U.S. Pat. No. 7,335,509,issued Feb. 26, 2008, which is a continuation of U.S. patent applicationSer. No. 09/376,395, filed on Aug. 18, 1999, now abandoned, which is acontinuation of U.S. patent application Ser. No. 08/939,874, filed onSep. 29, 1997, now U.S. Pat. No. 6,008,202, issued Dec. 28, 1999, whichis a continuation-in-part of U.S. patent application Ser. No.08/751,888, filed on Nov. 18, 1996, now abandoned, and acontinuation-in-part of U.S. patent application Ser. No. 08/376,701,filed on Jan. 23, 1995, now U.S. Pat. No. 5,795,587, issued Aug. 18,1998, the disclosures of each of which are hereby incorporated herein byreference in their entirety.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

This invention was made with Government support under Grant No. DK44935,awarded by the National Institutes of Health. The Government has certainrights in this invention.

FIELD OF INVENTION

The present invention relates to cationic lipids and their use asvehicles for the transfer of nucleic acids or other macromolecules, suchas proteins, into cells. More specifically, this invention relates tolipid-comprising drug delivery complexes which are stable, biologicallyactive, capable of being concentrated, and to methods for theirproduction.

BACKGROUND OF INVENTION

The development of new forms of therapeutics which use macromoleculessuch as proteins or nucleic acids as therapeutic agents has created aneed to develop new and effective means of delivering suchmacromolecules to their appropriate cellular targets. Therapeutics basedon either the use of specific polypeptide growth factors or specificgenes to replace or supplement absent or defective genes are examples oftherapeutics which may require such new delivery systems. Clinicalapplication of such therapies depends not only on the efficacy of newdelivery systems but also on their safety and on the ease with which thetechnologies underlying these systems can be adapted for large scalepharmaceutical production, storage, and distribution of the therapeuticformulations. Gene therapy has become an increasingly important mode oftreating various genetic disorders. The potential for providingeffective treatments, and even cures, has stimulated an intense effortto apply this technology to diseases for which there have been noeffective treatments. Recent progress in this area has indicated thatgene therapy may have a significant impact not only on the treatment ofsingle gene disorders, but also on other more complex diseases such ascancer. However, a significant obstacle in the attainment of efficientgene therapy has been the difficulty of designing new and effectivemeans of delivering therapeutic nucleic acids to cell targets. Thus, anideal vehicle for the delivery of exogenous genes into cells and tissuesshould be highly efficient in nucleic acid delivery, safe to use, easyto produce in large quantity and have sufficient stability to bepracticable as a pharmaceutical.

Non-viral vehicles, which are represented mainly by the cationicliposomes, are one type of vehicle which have, for the followingreasons, been considered for use in gene therapy. First, the plasmid DNArequired for liposome-mediated gene therapy can be widely and routinelyprepared on a large scale and is simpler and carries less risk than theuse of viral vectors such as retroviruses. Second, liposome-mediatedgene delivery, unlike retroviral-mediated gene delivery, can delivereither RNA or DNA. Thus, DNA, RNA, or an oligonucleotide can beintroduced directly into the cell. Moreover, cationic liposomes arenon-toxic, non-immunogenic and can therefore be used repeatedly in vivoas evidenced by the successful in vivo delivery of genes to catheterizedblood vessels (Nabel, E. G., et al. (1990) Science, 249: 1285-1288),lung epithelial cells (Brigham, K. L., et al. (1989) Am. J. Respir. CellMol. Biol., 195-200, Stribling, R., et al. (1992) Proc. Natl. Acad. Sci.U.S.A., 89: 11277-11281), and other systemic uses (Zhu, N., et al.(1993) Science, 261: 209-211, Philip, R., et al. (1993) Science, 261:209-211; Nabel, G. et al (1994) Hum. Gene Ther., 5:57-77) of cationicliposomes.

Although a variety of cationic liposome formulations, including thecommercially available cationic liposome reagent DOTMA/DOPE(N-1,-(2,3-dioleoyloxy) propyl-N,N,N-trimethyl ammoniumchloride/dioleoyl phosphatidylethanolamine), are known in the art(Felgner, P. L. et al. (1987) Proc. Natl. Acad. Sci. U.S.A., 84:7413-7417), a cationic liposome formulation designated DC-Chol/DOPE(3βN-(N′,N′-dimethylaminoethane)-carbamoyl cholesterol/(dioleoylphosphatidylethanolamine) has been shown in in vitro studies (Gao, X.,and Huang, L. (1991) Biochem. Biophys. Res. Commun., 179: 280-285) to berelatively non-toxic and more efficient than DOTMA/DOPE. Moreover,following extensive in vivo studies (Plautz, G. E., et al. (1993) Proc.Natl. Acad. Sci. U.S.A., 90: 4645-4649, Stewart, M. J., et al. (1992)Hum. Gene Ther., 3: 267-275) in which DC-Chol/DOPE was demonstrated tobe both safe and efficacious as a nucleic acid delivery system, thisformulation was approved by the U.S. Food and Drug Administration (FDA)and the U.K. Medicines Control Agency (MCA), and has been used in twoseparate gene therapy clinical trials (Nabel, G. J., et al. (1993) Proc.Natl. Acad. Sci. U.S.A., 90: 11307-11311, Caplen, N. J., et al. (1995)Nature Medicine, 1: 39-46).

However, the use of DC-Chol/DOPE and other currently existing cationicliposomes as vehicles for delivering nucleic acids to cellular targetsare inconvenient for large scale therapeutic applications for a numberof reasons. First, the ratios of liposome to nucleic acid utilized toform nucleic acid/liposome complex in the prior art admixture methodresults in the formation of complexes which are large in diameter andrelatively unstable. Thus, none of the presently utilized cationicliposome formulations, including DC-Chol/DOPE, are designed as stableand ready-to-use pharmaceutical formulations of nucleic acid/liposomecomplex. This limitation of the admixture method requires that the userprepare the complex prior to each use, an inconvenience which requiresspecial training of personnel. In addition, the preparation of thecomplex by admixture prior to each use introduces a possible source ofdosage variability which hinders evaluation of treatments utilizingthese complexes due to possible over- or under-dosing of the recipient.

Second, the prior art admixture method of preparing nucleicacid/cationic liposome complexes prior to each use requires that adilute nucleic acid solution (less than 4 μg/ml) and a dilute liposomedispersion (less than 50 μM) be used to prepare the nucleicacid/liposome complex in order to reduce the chance of forming large andless active aggregates. This limitation makes it difficult to make smallbiologically active complexes without using less than optimalconditions, such as reducing the amount of liposomes (which causesreduced nucleic acid transfer activity) or increasing the amount ofliposome (which causes enhanced toxicity). Moreover, the fact that thecomplex must be made in dilute concentrations is a significant drawbackto clinical applications, particularly in the case of intratumorinjection of the complex, since only a small volume of the complex canbe injected in each site (Nabel, G. J., et al. (1993) Proc. Natl. Acad.Sci. U.S.A., 90: 11307-11311).

Accordingly, an object of this invention is to provide stable,biologically active, lipid-comprising drug delivery complexes which arecapable of being formulated in a high concentration as well as methodsof producing such complexes.

SUMMARY OF INVENTION

This invention provides methods for producing lipid-comprising drugdelivery complexes having a net positive charge and/or a positivelycharged surface. By “drug” as used throughout the specification andclaims is meant any molecular entity, which is either monomeric oroligomeric, and which, when complexed with lipid or with lipid andpolycation, is being administered to an individual for the purpose ofproviding a therapeutic effect to the recipient. Thus, macromoleculeshaving an overall net negative charge or regions of negativity would beexpected to be capable of forming the delivery complexes of thisinvention. Macromolecules which are particularly suitable for use withthe complexes of this invention are for example, DNA, RNA,oligonucleotides or negatively charged proteins. However, macromoleculeshaving a positive charge (e.g., large cationic protein) would also beexpected to be capable of forming the complexes of this invention bysequentially complexing the cationic macromolecule with anionic moleculeor polymer and then with cationic lipid.

The complexes of the invention comprise a drug/lipid complex formed bymixing the drug to be delivered with cationic liposomes in a drug tolipid ratio such that the drug/lipid complex formed has a net positivecharge and a drug/lipid/polycation complex formed by mixing drug withcationic liposomes and polycation in a drug to lipid to polycation ratiosuch that the drug/lipid/polycation complex formed has a net positivecharge. By “net positive charge” as applied to the drug/lipid complex ismeant a positive charge excess of lipid to drug. By “net positivecharge” as applied to the drug/lipid/polycation complex is meant thatthe positive charges of the cationic lipid and the polycation exceed thenegative charge of the drug. However, it is to be understood that thepresent invention also encompasses drug/lipid and drug/lipid/polycationcomplexes having a positively charged surface irrespective of whetherthe net charge of the complex is positive, neutral or even negative. Apositively charged surface of a complex may be measured by the migrationof the complex in an electric field by methods known to those in the artsuch as by measuring zeta potential (Martin, A., Swarick, J., andCammarata, A., Physical Pharmacy & Physical Chemical Principles in thePharmaceutical Sciences, 3rd ed. Lea and Febiger, Philadelphia, 1983),or by the binding affinity of the complex to cell surfaces. Complexesexhibiting a positively charged surface have a greater binding affinityto cell surfaces than complexes having a neutral or negatively chargedsurface. Furthermore, the positively charged surface could be stericallyshielded by the addition of non-ionic polar compounds, of whichpolyethylene glycol is an example.

The invention therefore relates to methods for producing thesedrug/lipid and drug/lipid/polycation complexes comprising mixing thedrug to be delivered with cationic liposomes, and optionally polycation,in a ratio such that the complex formed has a net positive charge and/ora positively charged surface.

In another embodiment of this invention, the methods for producing druglipid or drug/lipid/polycation complexes may further comprise the stepof purifying said complexes from excess free components (drug, lipid,polycation) following their production.

The drug/lipid and drug/lipid/polycation complexes of this invention aregenerally stable, capable of being produced at relatively highconcentration, and retain biological activity over time in storage. Suchcomplexes are of utility in the delivery of nucleic acids, proteins andother macromolecules to cells and tissues.

In another embodiment of this invention complexes are found comprisingpolycationic polypeptides having a high arginine content.

DESCRIPTION OF FIGURES

FIG. 1 shows a typical size distribution (mean diameter) of nucleicacid/liposome complexes prepared as an admixture from DC-Chol/DOPE (3:2mol/mol) liposomes and pRSVL plasmid DNA (2 μg) at the indicated lipidto DNA ratios.

FIGS. 2A and 2B show the distribution of the liposome marker³H-cholesteryl hexadecyl ether (◯) and the ³²P-DNA marker (▪) amongsucrose gradient fractions. The location of each fraction in the sucrosegradients of both FIGS. 2A and 2B is indicated at the top of FIG. 2A.

FIG. 2A shows the distribution of the ³H and ³²P markers followingultracentrifugation of free liposomes (10 μmoles of DC-Chol/DOPE (2:3)in 2 ml volume) or free DNA (50 μg pRSVL DNA in a 2 ml volume) through asucrose density gradient.

FIG. 2B shows the distribution of the ³H and ³²P markers followingultracentrifugation of the DNA-lipid complex (formed via mixing of 20μmoles DC-Chol/DOPE (2:3) liposomes and 0.4 mg pRSVL DNA in 2 ml volume)through a sucrose density gradient.

FIG. 3 shows the transfection activities in CHO cells of admixtureDNA/liposome complex (◯), admixture DNA/liposome/poly-L-lysine(PLL)complex (□) DNA/lipid complex (•) and DNA/lipid/PLL complex (▪). TheDC-Chol/DOPE liposomes used to form the above complexes containedvarying mole % of DC-Chol as indicated at the bottom of FIG. 3. TheDNA/lipid (•) and DNA/lipid/PLL (▪) complexes were purified on a sucrosedensity gradient prior to being assayed for transfection activity.Transfection activity is indicated on the vertical axis as relativelight units of luciferase activity.

FIG. 4 shows the transfection activities of admixture DNA/liposomecomplex (◯) and admixture DNA/liposome/PLL complex (□) compared to thetransfection activities of DNA/lipid (•) and DNA/lipid/PLL complexesstored at 4° C. for 130 days following their purification on a sucrosedensity gradient. The DC-Chol/DOPE liposomes used to form the abovecomplexes contained varying mole % of DC-Chol as indicated at the bottomof FIG. 4. Transfection activity is indicated on the vertical axis asrelative light units of luciferase activity.

FIG. 5 shows the concentration of extractable protein from CHO cells, 36hours after the cells were treated with admixture DNA/liposome complex(◯); admixture DNA/liposome/PLL complex (▪); DNA/lipid complex (•); orDNA/lipid/PLL complex (▪). The DNA/lipid and DNA/lipid/PLL complexeswere purified on a sucrose density gradient prior to being assayed fortransfection activity. The DC-Chol/DOPE liposomes used to form the abovecomplexes contained varying mole % of DC-Chol as indicated at the bottomof FIG. 5.

FIG. 6 shows the results of CAT assays of tumor extracts prepared frommice having ovarian tumors. 2×10⁶ human ovarian carcinoma cells weresubcutaneously injected into SCID mice at day 0. On day 14, 100 μl Asolutions containing pUCCMVCAT DNA (contains the chloramphenicol acetyltransferase gene of E. coli) 30 μg) complexed with DC-Chol liposomes (30nmoles) in the form of admixture (lanes 1 and 2; duplicate samples) orthe same amount of DNA in the form of purified complex (prepared fromDNA:DC-chol liposome at ratio 1 μg/25 nmoles, lanes 3 and 4; duplicatesamples) were directly injected into tumors. 48 hours followingtransfection, the mice were sacrificed and tumor extract containing 100μg protein was assayed for CAT activity. Lane 5 shows positive controlCAT activity for standard E. coli CAT.

FIGS. 7A-7C show the transfection activities of admixture DNA/lipidcomplex and purified and unpurified DNA/lipid/PLL complexes in 293 cells(FIG. 7A) C3 cells (FIG. 7B) and BL6 cells (FIG. 7C). Transfectionactivity is indicated on the vertical axis of FIGS. 7A-7C as relativelight units of luciferase activity.

FIGS. 8A and 8B show a comparison of the ability of protamine sulfate,USP (PS) and poly-L-Lysine (PLL) to increase the transfection activityin CHO and 293 cells, respectively. Varying amounts of PS (•) or PLL (◯)were added to 1 μg of pUK-21 CMV LUC DNA prior to complexation, with 7.5nmol of DC-Chol liposomes per well. Transfection, luciferase, andprotein assays were performed as described in Example 9 and each datapoint represents the mean (with standard variation) of three data pointsand are normalized to protein content.

FIG. 9 shows the effect on transfection activity of different types ofprotamine where 2 μg of each indicated type of protamine or 1 μgpoly-L-lysine were added to 1 μg of pUK-21 CMV LUC DNA prior tocomplexing with 7.5 nmol of DC-Chol liposomes per well. Transfection,luciferase, and protein assays were performed as described in Example 10and each point represents the mean (with standard deviation) of fourdata points and are normalized to protein.

FIG. 10 shows two different formulations which were prepared by mixingpCMV-Luc DNA with DOTAP (25.38 μg/μg DNA) or DNA with Protamine Sulfate,USP (PS) and complexing this mixture with DOTAP (0.8 μPS/μg DNA/23.27 μgDOTAP). Dextrose was added to both formulations to make a finalconcentration of 5%, and were subsequently injected into mice (CD 1female, 4-6 weeks old) through the tail vein (50 μg of DNA/mouse).Twenty-four hours following injection, the mice were sacrificed and themajor organs collected. Tissues were homogenized in a lysis buffer,centrifuged at 14,000 g for 10 min and the supernatant was analyzed forluciferase activity and protein content. The results are expressed asrelative light units (RLU) per mg of protein (n=3). No activity wasfound with DNA complexed with PS (0.8 μg PS/μg DNA) in the absence ofDOTAP (data not shown). Complexation of DOTAP with DNA gave a variablelevel of expression, mainly in the lung. Inclusion of Protamine Sulfate,USP into the DNA/DOTAP complex resulted in a more consistent and higherlevel of gene expression.

FIG. 11 shows in vivo gene expression of PS/DNA/DOTAP complexes as afunction of DOTAP concentration. DNA was mixed with PS (0.8 μg PS/μgDNA) followed by complexation with increasing amounts of DOTAP.Injection of these complexes and assay of gene expression were performedas described in FIG. 10. The results indicate increasing amounts ofDOTAP were associated with increases in gene expression in the lungs andspleen (n=3).

FIG. 12 shows in vivo gene expression of PS/DNA/DOTAP complexes as afunction of the dose of the complex delivered. DNA was mixed with PSfollowed by complexation with DOTAP (0.8 μg PS/μg DNA/23.27 μg DOTAP) atincreasing doses, as expressed by the dose of DNA. Increasing doses ofthe complex were injected into mice and gene expression was assayed 24hours later (n=3). The results indicate increasing doses of thePS/DNA/DOTAP complex were associated with an increase in geneexpression. However, at the dose above 75 μg (of DNA) per mouse,toxicity was noticed and one mouse died at a dose of 100 μg (of DNA).

FIG. 13 shows in vivo gene expression of PS/DNA/DOTAP complexes as afunction of time. PS/DNA/DOTAP complexes were prepared as described inFIG. 12 and were injected into mice at a DNA dose of 50 μg per mouse. Atdifferent times following injection, the mice were sacrificed and themajor organs were assayed for gene expression (n=3). It can be seen thathigh levels of gene expression was observed in the lung as early as 6hours following injection and persisted for 24 hours, decliningthereafter. High levels of gene expression were also detected in thespleen 6 hours after injection but declined with time.

FIG. 14 shows the in vitro transfection activity of increasing amountsof PS/DNA (2:1 w/w ratio) at a fixed amount (7.5 nmol) of DC-Cholliposomes in CHO cells. Optimal activity was seen at a dose of 2 μg ofDNA, 4 μg PS and 7.5 nmol of DC-Chol liposomes.

FIG. 15 shows the in vitro transfection activity of increasing amountsof DC-Chol liposomes at a fixed amount of PS/DNA (2 μg PS; 1 μg DNA) inCHO cells. Optimal activity was seen at 2.5 nmol of DC-Chol liposomesand remained constant thereafter, indicating that 2.5 nmol of DC-Cholliposomes was sufficient to adequately deliver 2 μg of PS and 1 μg ofDNA.

FIGS. 16-19 show the ability of PS to augment gene expression whencomplexed to DNA with Clonfectin (Clontech) (FIG. 16), DC-Chol liposomes(FIG. 17), Lipofectin (Gibco BRL) (FIG. 18), and DOTAP/DOPE (1:1 m/m)(Avanti Polar Lipids) (FIG. 19) formulations. CHO cells were transfectedwith various amounts of liposomes complexed with 1 μg of DNA with (▪) orwithout (□) the inclusion of 2 μg of PS. With each liposome formulation,significant increases in transfection activity are noted in complexescontaining PS when compared to complexes consisting of DNA alone withlipid. These results indicate that the increase in transfection activityis not limited to DC-Chol liposomes.

FIG. 20 shows the relationship between Lipid:Protamine Sulfate:DNAratios and particle sizes. The X axis describes the ProtamineSulfate:DNA ratio. The Y axis describes the Lipid:DNA ratio. Particlesizes (nm) corresponding to the respective ratios at Day 0 and Day 7appear in the matrix cell at the intersection of the X and Y axes.Particle sizes around 200 nm are thought to be favorable since thisrepresents optimal sizing for coated pit internalization.

FIG. 21 shows the in vitro transfection activities of different LPDformulations in HeLa cells (a human cervical carcinoma cell line). TheProtamine sulfate concentration of each formulation used is shown on theX axis. Transfection activity is indicated on the Y axis as relativelight units of luciferase activity normalized to the amount of extractedprotein.

FIG. 22 shows the in vitro transfection activities of different LPDformulations in SKOV-3 cells (a human ovarian carcinoma cell line). Thevarious DC-Chol and LPD delivery vehicles used in the study areindicated on the X axis. Transfection activity is indicated on the Yaxis as relative light units of luciferase activity normalized to theamounts of extracted protein. The n value represents the number ofindividual experiments which were averaged for the data table. Eachindividual experiment consisted of replicates where n=3 or 6 for eachformulation represented.

FIG. 23 shows the relationship between the Lipid:Protamine Sulfate:DNAformulation used in vivo to deliver pCMV-Luc intraperitoneally in anSKOV-3 murine model and the observed Luciferase expression activity. Thecomposition of the delivery formulation is indicated on the X axis. Thenomenclature used to describe the formulations follows the pattern ofnanomoles of Lipid:μg Protamine Sulfate:μg DNA. Transfection activity isindicated on the Y axis as relative light units of luciferase activity.The graph shows the RLU/mg values obtained for each individual mouse(circles) and the average value per formulation (squares).

FIG. 24 shows a dose response graph for the treatment of Nude miceinjected intraperitoneally with 2×10⁶ SKOV-3ip1 cells at Day 0. On Day 5mice were injected with different L:P:D formulations comprising an E1Aexpression plasmid. Group 1 animals were repeatedly injected with the 5%Dextrose vehicle only; Group 2 animals were repeatedly injected with theequivalent amount of DC-Chol/DOPE (6:4 mol:mol ratio) and Protaminesulfate as one would find in the high dose 15:2:1 group but without theE1A expression plasmid present; Group 3 animals were repeatedly injectedwith 15 μg/dose of naked E1A expression plasmid, which was equivalent tothe highest dose injected in formulated vehicles; Group 4 animals wererepeatedly injected with 15 μg/dose of E1A expression plasmid compactedwith Protamine sulfate at a ratio of 2 μg Protamine/1 μg DNA, which wasequivalent to the highest dose injected in the formulated vehicles;Group 14 animals were repeatedly injected with a 1.5 μg/dose 15:2:1 LPDformulation; Group 15 animals were repeatedly injected with a 5 μg/dose15:2:1 LPD formulation; Group 16 animals were repeatedly injected with a15 μg/dose LPD formulation. The time course of the experiment in days isshown on the X axis. The number of surviving mice is shown on the Yaxis.

DESCRIPTION OF INVENTION

This invention relates to lipid-comprising drug delivery complexeshaving a net positive charge and/or a positively charged surface at pH6.0-8.0. These complexes comprise lipids, drugs, and optionally furthercomprise polycations. The invention further relates to a method forproducing these complexes where the method may optionally include thestep of purifying these formulations from excess individual components.For the production of the drug/lipid complexes of this invention,inclusion of the purification step is a preferred embodiment. It shouldbe understood that where the purification step is applied to thedrug/lipid/polycation complexes, the recovery of these complexes in apure state free from excess components following purification is lowerthan the recovery of drug/lipid complexes following their purificationsince the peak containing the drug/lipid/polycation complex followingsucrose purification via density centrifugation is broader than the peakcontaining drug/lipid complexes and hence, overlaps with the peaks ofthe free components.

The lipid-comprising drug delivery complexes of this invention arestable, capable of being produced at relatively high concentrations, andretain biological activity of the drug component over time in storage.The method of producing these complexes is based on a binding modelbetween two oppositely charged polymers (e.g. negatively charged nucleicacid and positively charged lipids) in which the formation of largeunstable aggregates is avoided by neutralizing the negative charge ofthe drug via the use of an excess amount of positive charge in the formof cationic liposomes or cationic liposomes and polycation. Thecomplexes of this invention have been observed to retain their initialdiameter and bioactivity over 4 months in storage in 10% sucrose buffer.

The “drug” which is contained in the lipid-comprising drug deliverycomplexes of the present invention may be nucleic acids, polyanionicproteins, polysaccharides and other macromolecules which can becomplexed directly with cationic lipids. However, cationic drugs (eglarge cationic protein) can be directly complexed with an anionic lipidor sequentially complexed first with anionic lipid or polymer followedby cationic lipid. The use of this process permits delivery of positiveor neutral charged drug to cells by the complexes of the presentinvention.

To produce drug/lipid and drug/lipid/polycation complexes with a netpositive charge, the positive charge excess of lipid to drug or of lipidand polycation to drug may be up to about a 30-fold positive chargeexcess in the complex of total lipids to drug or of lipid and polycationto drug, preferably about a 2 to 10-fold charge excess and mostpreferably about a 2 to 6-fold charge excess. Complexes which possess apositive charge on their surface may have similar preferred ranges ofsurface charge excess to drug. To produce a nucleic acid/lipid complexhaving a positive charge excess of lipid to nucleic acid, mole amountsof cationic liposomal lipid to be mixed with 1 μg of nucleic acid toproduce a nucleic acid/lipid complex which has positive charge excess oflipid to nucleic acid at pH 6.0-8.0 may range from about 0.1 nmol toabout 200 nmol of lipid, preferably about 5 nmol to about 100 nmollipid, depending on the positive charge content of the cationicliposome. Of course, if the drug were a protein, the amount of lipid tobe mixed with 1 μg of negatively charged protein would be at least10-fold less than the amount of lipid to be mixed with 1 μg of DNA asshown above since proteins are less charge dense than nucleic acids.Those of ordinary skill in the art would readily understand thatdepending upon the positive charge content of the cationic liposomes,different mole amounts of different cationic liposomes would have to bemixed with an equivalent amount of drug to produce a positive chargeexcess of lipid to drug.

When a drug/lipid/polycation complex having a net positive charge and/ora positively charged surface is to be produced, the inclusion of thepolycation reduces the amount of lipid which must be mixed with drug tothe extent that the positive charge from the lipid may be less than thenegative charge from the drug. This reduction in the amount of lipidreduces the toxicity of the polycation-containing formulations. Moleamounts of cationic liposomes to be used in formulating nucleicacid/lipid/polycation complexes may range from about 0.1 nmol to about200 nmol lipid per 1 μg nucleic acid, more preferably from about 1 toabout 25 nmoles lipid per 1 μg nucleic acid depending on the positivecharge content of the cationic liposomes. It is to be generallyunderstood that in producing the nucleic acid/lipid and nucleicacid/lipid/polycation complexes of the present invention, the moleamount of liposomes required to produce these complexes will increase asthe concentration of nucleic acid mixed with the liposomes is increased.

Those of ordinary skill in the art would readily understand that whenthe complexes of the present invention are purified, the positive chargeexcess of cationic liposomes to drug or of cationic liposomes andpolycation to drug immediately prior to mixing will be greater than thepositive charge excess in the purified complexes of lipid to drug or oflipid and polycation since the purification step results in the removalof excess free lipids and/or free polycation.

In order to illustrate how the charges attributed to cationic lipid,drug and polycation may be determined at pH 6.0-8.0 the followingexample is provided. Assuming the drug to be delivered is DNA, onedetermines the negative charge of the DNA to be delivered by dividingthe amount of DNA to be mixed, or the amount of DNA in the complex, by330, the molecular weight of a single nucleotide where one nucleotideequals one negative charge. Thus, the negative charge for 1 μg of DNA is3.3 nmols.

For 10 nmol of DC-Chol/DOPE (2:3) liposomes one calculates the effectivecharge of the lipid by multiplying the amount of total liposomal lipid(10 nmol) by 0.4 (40% of the total liposomal lipid is the cationic lipidDC-Chol) to yield 4 nmol DC-Chol lipid in the liposomes. Since at pH6-8, one molecule of DC-Chol has one positive charge, the effectivepositive charge of liposomal lipid at the time of mixing, or in thecomplex, is 4.0 nmol. Of course, those of skill in the art would readilyunderstand that other cationic lipids may have a lesser or greateramount of positive charge per molecule of cationic lipid at pH 6-8.0than DC-Chol.

Assuming the polycation to be mixed to form the complex is a brominesalt of poly-L-lysine (PLL), the positive charge of PLL at the time ofmixing is obtained by dividing the amount of PLL to be mixed by 207, themolecular weight of one lysyl residue where one lysyl residue equals onepositive charge. Thus, the positive charge for 1 μg of PLL isapproximately 5.0 nmols. To calculate the positive charge contributed bylysyl residues in a formed complex, the amount of lysine present in thecomplex is divided by the molecular weight of one lysyl residue takinginto account the weight of a counterion, if present.

Application of the above calculations to data presented in Table 1herein (see Example 3) illustrates how a positive to negative chargeratio can be calculated both at the time of mixing of DNA and liposomeand, after purification of the complex produced by the mixing of DNA andliposome. In Table 1 of Example 3, 0.4 mg of DNA is mixed with 20 μmolsof cationic DC-Chol/DOPE liposomes to produce DNA/lipid complex. Forcationic liposomes having a DC-Chol/DOPE ratio of 4:6, the positivecharge content of the liposomal lipid is calculated to be 8000 nmol andthe negative charge content of the 0.4 mg DNA to be mixed with liposomesis calculated to be 1320 nmols based on the sample calculationspresented in the above paragraphs. Therefore, the positive to negativecharge ratio at the time of mixing is 6.06 (8000 divided by 1320).However, after the complex was purified, the lipid to DNA ratio of thispurified complex was 12.7 nmol lipid/μg DNA as shown in Table 1 (see the“4:6 row”). This 12.7 ratio translates to a positive to negative chargeratio of 1.5 thus showing that purification removed excess positivecharge of free liposomes.

Also in Table 1, where DNA/lipid/PLL complex was prepared by mixing 4μmol of liposomes (4:6 DC-Chol/DOPE) and 1 mg PLL with 0.4 mg DNA, onecan calculate the positive to negative charge ratio at the time ofmixing as follows. Based on the sample calculations presented in theabove paragraphs, the 4 μmol liposomal lipid contributes 1600 nmol ofpositive charge, the 1 mg of PLL contributes 5000 nmol of positivecharge and the 0.4 mg DNA contributes 1,320 nmol of negative charge.Thus, the positive to negative charge ratio at the time of mixing

${liposomes},\mspace{14mu}{{PLL}\mspace{14mu}{and}\mspace{14mu}{DNA}\mspace{14mu}{is}\mspace{14mu} 5{\frac{\left( {1600 + 5000} \right)}{1320}.}}$

It is further to be understood by those skilled in the art that the netcharge of the complex may be determined by measuring the amount of DNA,lipid and when present, polycation in the complex by the use of anappropriate analytical technique such as the use of radioisotopiclabelling of each component or by elemental analysis. Once the amountsof each component (DNA, lipid and when present, polycation) in a complexat a given pH are known, one could then calculate the approximate netcharge of that complex at the given pH taking into account the pK's ofthe components which may be known or determined analytically.

In a preferred embodiment, the drug is a nucleic acid sequence,preferably a nucleic acid sequence encoding a gene product havingtherapeutic utility.

In one embodiment of the invention, a method for producing nucleicacid/lipid complexes having a net positive charge and/or positivelycharged surface at pH 6-8.0, comprises, combining nucleic acids withcationic liposomes in a nucleic acid to lipid ratio such that thenucleic acid/lipid complex formed has a positive charge excess of lipidto nucleic acid.

In an alternative embodiment, nucleic acid and cationic liposome may bemixed with a polycation in a nucleic acid to lipid to polycation ratiosuch that the nucleic acid/lipid/polycation complexes formed have apositive charge excess of lipid and polycation to nucleic acid at pH6-8.

In a preferred embodiment, the nucleic acid/lipid and nucleicacid/lipid/polycation complexes are produced by slowly adding nucleicacid to the solution of liposome or liposome plus polycation and mixingwith a stirring bar where the mixing is allowed to proceed second afteraddition of DNA. Alternatively, the liposome or liposome/polycation mixcan be added into a single chamber from a first inlet at the same timethe nucleic acid is added to the chamber through a second inlet. Thecomponents are then simultaneously mixed by mechanical means in a commonchamber. The complexes may also be produced by first mixing the nucleicacid with the polycation and then adding the liposome suspension.

The cationic liposomes mixed with drug or with drug and polycation toform the complexes of the present invention may contain a cationic lipidalone or a cationic lipid in combination with a neutral lipid. Suitablecationic lipid species include, but are not limited to:3β[⁴N-(¹N,⁸N-diguanidino spermidine)-carbamoyl]cholesterol (BGSC);3β[N,N-diguanidinoethyl-aminoethane)-carbamoyl]cholesterol (BGTC);N,N¹,N²,N³ Tetra-methyltetrapalmitylspermine (cellfectin);N-t-butyl-N′-tetradecyl-3-tetradecyl-aminopropion-amidine (CLONfectin);dimethyldioctadecyl ammonium bromide (DDAB);1,2-dimyristyloxypropyl-3-dimethyl-hydroxy ethyl ammonium bromide(DMRIE);2,3-dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluorocetate) (DOSPA); 1,3-dioleoyloxy-2-(6-carboxyspermyl)-propylamide (DOSPER); 4-(2,3-bis-palmitoyloxy-propyl)-1-methyl-1H-imidazole(DPIM) N,N,N′,N′-tetramethyl-N,N′-bis(2-hydroxyethyl)-2,3dioleoyloxy-1,4-butanediammonium iodide) (Tfx-50); 1,2bis(oleoyloxy)-3-(trimethylammonio) propane (DOTAP);N-1-(2,3-dioleoyloxy) propyl-N,N,N-trimethyl ammonium chloride (DOTMA)or other N—(N,N-1-dialkoxy)-alkyl-N,N,N-trisubstituted ammoniumsurfactants; 1, 2 dioleoyl-3-(4′-trimethylammonio) butanol-sn-glycerol(DOBT) or cholesteryl (4′ trimethylammonia) butanoate (ChOTB) where thetrimethylammonium group is connected via a butanol spacer arm to eitherthe double chain (for DOTB) or cholesteryl group (for ChOTB); DORI(DL-1,2-dioleoyl-3-dimethylaminopropyl-(3-hydroxyethylammonium) or DORIE(DL-1,2-O-dioleoyl-3-dimethylaminopropyl-(3-hydroxyethylammonium)(DORIE) or analogs thereof as disclosed in WO 93/03709;1,2-dioleoyl-3-succinyl-sn-glycerol choline ester (DOSC); cholesterylhemisuccinate ester (ChOSC); lipopolyamines such asdioctadecylamidoglycylspermine (DOGS) and dipalmitoylphosphatidylethanolamylspermine (DPPES) or the cationic lipids disclosedin U.S. Pat. No. 5,283,185,cholesteryl-3(3-carboxyl-amido-ethylenetrimethyl-ammonium iodide,1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesteryl carboxylateiodide, cholesteryl-3β-carboxyamidoethyleneamine,cholesteryl-3β-oxysuccinamido-ethylenetrimethylammonium iodide,1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesteryl-3β-oxysuccinateiodide, 2-(2-trimethylammonio)-ethylmethylaminoethyl-cholesteryl-3β-oxysuccinate iodide,3β-N—(N′,N′-dimethylaminoethane) carbamoyl cholesterol (DC-chol), and3β-N-(polyethyleneimine)-carbamoylcholesterol.

Examples of preferred cationic lipids includeN-t-butyl-N′-tetradecyl-3-tetradecyl-aminopropion-amidine (CLONfectin),2,3-dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoroacetate (DOSPA), 1,2-bis(oleoyloxy)-3-(trimethylammonio)propane(DOTAP), N-[1-(2,3,dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride)(DOTMA), cholesteryl-3β-carboxyamidoethylenetri-methylammonium iodide,1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesteryl carboxylateiodide, cholesteryl-3β-carboxyamidoethyleneamine,cholesteryl-3β-oxysuccin-amidoethylenetrimethyl-ammonium iodide,1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesteryl-3β-oxysuccinateiodide, 2-(2-trimethylammonio)ethylmethylaminoethyl-cholesteryl-3β-oxysuccinateiodide, 3βN—(N′,N′dimethyl-aminoethane)-carbamoyl-cholesterol (DC-chol), and3βN-(polyethyleneimine)-carbamoyl cholesterol.

Since an attribute of the complexes of the invention is their stabilityduring storage (i.e., their ability to maintain a small diameter andretain biological activity over time following their formation); it willbe understood by those of ordinary skill in the art that preferredcationic lipids are those lipids in which bonds between the lipophilicgroup and the amino group are stable in aqueous solution. While suchbonds found in cationic lipids include amide bonds, ester bonds, etherbonds and carbamoyl bonds, preferred cationic lipids are those having acarbamoyl bond. An example of a preferred cationic lipid having acarbamoyl bond is DC-Chol. Those of skill in the art would readilyunderstand that liposomes containing more than one cationic lipidspecies may be used to produce the complexes of the present invention.For example, liposomes comprising two cationic lipid species,lysyl-phosphatidylethanolamine and β-alanyl cholesterol ester have beendisclosed (Brunette, E. et al. (1992) Nucl. Acids Res., 20:1151).

It is to be further understood that in considering cationic liposomessuitable for use in mixing with drug and optionally with polycation, toform the complexes of this invention, the methods of the invention arenot restricted only to the use of the lipids recited above but rather,any lipid composition may be used so long as a cationic liposome isproduced.

Thus, in addition to cationic lipids, cationic liposomes used to formthe complexes of the invention may contain other lipids in addition tothe cationic lipids. These lipids include, but are not limited to, lysolipids of which lysophosphatidylcholine (1-oleoyllysophosphatidylcholine) is an example, cholesterol, or neutralphospholipids including dioleoyl phosphatidyl ethanolamine (DOPE) ordioleoyl phosphatidylcholine (DOPC) as well as various lipophilicsurfactants, containing polyethylene glycol moieties, of which Tween-80is one example. The lipid complexes of the invention may also containnegatively charged lipids as well as cationic lipids so long as the netcharge of the complexes formed is positive and/or the surface of thecomplex is positively charged. Negatively charged lipids of theinvention are those comprising at least one lipid species having a netnegative charge at or near physiological pH or combinations of these.Suitable negatively charged lipid species include, but are not limitedto, CHEMS (cholesteryl hemisuccinate), NGPE (N-glutarylphosphatidlylethanolanine), phosphatidyl glycerol and phosphatidic acidor a similar phospholipid analog.

It is further contemplated that in the cationic liposomes utilized toform the complexes of the invention, the ratio of lipids may be variedto include a majority of cationic lipids in combination with cholesterolor with mixtures of lyso or neutral lipids. When the cationic lipid ofchoice is to be combined with another lipid, a preferred lipid is aneutral phospholipid, most preferably DOPE.

Methods for producing the liposomes to be used in the production of thelipid-comprising drug delivery complexes of the present invention areknown to those of ordinary skill in the art. A review of methodologiesof liposome preparation may be found in Liposome Technology (CFC PressNY 1984); Liposomes by Ostro (Marcel Dekker, 1987); Methods BiochemAnal. 33:337-462 (1988) and U.S. Pat. No. 5,283,185. Such methodsinclude freeze-thaw extrusion and sonication. Both unilamellar liposomes(less than about 200 nm in average diameter) and multilamellar liposomes(greater than about 300 nm in average diameter) may be used as startingcomponents to produce the complexes of this invention.

In the cationic liposomes utilized to produce the drug/lipid complexesof this invention, the cationic lipid is present in the liposome at fromabout 10 to about 100 mole % of total liposomal lipid, preferably fromabout 20 to about 80 mole % and most preferably about 20 to about 60mole %. The neutral lipid, when included in the liposome, may be presentat a concentration of from about 0 to about 90 mole % of the totalliposomal lipid, preferably from about 20 to about 80 mole %, and mostpreferably from 40 to 80 mole %. The negatively charged lipid, whenincluded in the liposome, may be present at a concentration ranging fromabout 0 mole % to about 49 mole % of the total liposomal lipid,preferably from about 0 mole % to about 40 mole %. In a preferredembodiment, the liposomes contain a cationic and a neutral lipid, mostpreferably DC-Chol and DOPE in ratios between about 2:8 to about 6:4. Itis further understood that the complexes of the present invention maycontain modified lipids, protein, polycations or receptor ligands whichfunction as a targeting factor directing the complex to a particulartissue or cell type. Examples of targeting factors include, but are notlimited to, asialoglycoprotein, insulin, low density lipoprotein (LDL),folate and monoclonal and polyclonal antibodies directed against cellsurface molecules. Potential targets include, but are not limited to,liver, blood cells, endothelial cells and tumor cells. Furthermore, toenhance the circulatory half-life of the complexes, the positive surfacecharge can be sterically shielded by incorporating lipophilicsurfactants which contain polyethylene glycol moieties.

It is to be further understood that the positive charge of the complexesof this invention may be affected not only by the lipid composition ofthe complex but also by the pH of the solution in which the drug/lipidcomplexes are formed. For example, increasing pH (more basic) willgradually neutralize the positive charge of the tertiary amine of thecationic lipid DC-Chol. In a preferred embodiment, the complexes of thepresent invention are produced, and stored, at a pH such that thecomplexes have a net positive charge and/or positively charged surface.A preferred pH range is pH 6.0-8.0, most preferably pH 7.0-7.8.

When a polycation is to be mixed with nucleic acid and cationicliposomes, the polycation may be selected from organic polycationshaving a molecular weight of between about 300 and about 200,000. Thesepolycations also preferably have a valence of between about 3 and about1000 at pH 7.0. The polycations may be natural or synthetic amino acids,peptides, proteins, polyamines, carbohydrates and any synthetic cationicpolymers. Nonlimiting examples of polycations include polyarginine,polyornithine, protamines and polylysine, polybrene (hexadimethrinebromide), histone, cationic dendrimer, spermine, spermidine andsynthetic polypeptides derived from SV40 large T antigen which hasexcess positive charges and represents a nuclear localization signal. Inone embodiment, the polycation is poly-L-lysine (PLL).

In another more preferred embodiment, the polycation is a polycationicpolypeptide having an amino acid composition in which arginine residuescomprise at least 30% of the amino acid residues of the polypeptide andlysine residues comprise less than 5% of the amino acid residues of thepolypeptide. In addition, preferably histidine, lysine and argininetogether make up from about 45% to about 85% of the amino acid residuesof the polypeptide and serine, threonine and glycine make up from about10% to about 25% of the amino acid residues of the polypeptide. Morepreferably, arginine residues constitute from about 65% to about 75% ofthe amino acid residues of the polypeptide and lysine residuesconstitute from about 0 to about 3% of the amino acid residues of thepolypeptide.

In addition to the above recited percentages of arginine and lysineresidues, the polycationic polypeptides of the invention may alsocontain from about 20% to about 30% hydrophobic residues, morepreferably, about 25% hydrophobic residues. The polycationic polypeptideto be used in producing drug/lipid/polycation complexes may be up to 500amino acids in length, preferably about 20 to about 100 amino acids inlength; more preferably, from about 25 to about 50 amino acids inlength, and most preferably from about 25 to about 35 amino acids inlength.

In one embodiment, the arginine residues present in the polycationicpolypeptide are found in clusters of 3-8 contiguous arginine residuesand more preferably in clusters of 4-6 contiguous arginine residues.

In another embodiment, the polycationic polypeptide is about 25 to about35 amino acids in length and about 65 to about 70% of its residues arearginine residues and 0 to 3% of its residues are lysine residues.

The polycationic polypeptides to be used in formulating the complexes ofthe invention may be provided as naturally occurring proteins,particularly certain protamines having a high arginine to lysine ratioas discussed above, as a chemically synthesized polypeptide, as arecombinant polypeptide expressed from a nucleic acid sequence whichencodes the polypeptide, or as a salt of any of the above polypeptideswhere such salts include, but are not limited to, phosphate, chlorideand sulfate salts.

In one embodiment, a drug such as DNA could be complexed with an excessof polycationic polypeptide such that a net positively charged complexis produced. This complex, by nature of its positive charge, couldfavorably interact with negatively charged lipid (s) to form aDNA/lipid/polycationic polypeptide complex.

The transfection activity of a polycationic polypeptide/DNA/lipidcomplex of the invention in CHO cells, is preferably equal to or greaterthan the transfection activity of a poly-L-lysine/DNA/lipid complex inCHO cells when each polycation is complexed with the same cationicliposome and plasmid construct containing a reporter gene where reportergenes include, but are not limited to, the chloramphenicol acetyltransferase gene, the luciferase gene, the β-galactosidase gene and thehuman growth hormone gene, the alkaline phosphatase gene and a greenfluorescent protein gene.

In producing nucleic acid/lipid/polycation complexes of the presentinvention, the ratio of polycation to nucleic acid is kept fixed whilevarying the amount of liposome. However, those of skill in the art wouldrecognize that the ratio of polycation to nucleic acid will be affectedby the charge density of the liposome to be mixed with the nucleic acidand polycation. For example, if the charge density of liposomes isdecreased as a result of changes in the lipid composition of theliposome (eg decreasing the ratio of cationic lipid:neutral lipid in theliposome), the amount of polycation to be mixed with nucleic acid andliposome may be increased to compensate for the decrease in positivecharge contributed by the liposomes. However, when polycation isutilized, it is preferred to use subsaturating amounts of polycation(i.e., amounts which will not saturate all the negative charge of thenucleic acid) in order to allow the cationic lipids to complex with thenucleic acid. Thus, in a preferred embodiment of the invention, apositive charge excess of lipid to nucleic acid is used even whenpolycation is mixed with lipid and nucleic acid. Amounts of polycationwhich may be mixed with 1 μg of nucleic acid and varying amounts ofcationic liposomes in the present invention range from about 0.01 μg toabout 100 μg of polycation per μg of nucleic acid, preferably from about0.1 μg to about 10 μg of polycation per μg of nucleic acid.

Where purification of nucleic acid/lipid and nucleicacid/lipid/polycation complexes from excess free DNA, free liposomes andfree polycation is desired, purification may be accomplished bycentrifugation through a sucrose density gradient or other media whichis suitable to form a density gradient. However, it is understood thatother methods of purification such as chromatography, filtration, phasepartition, precipitation or absorption may also be utilized. In apreferred method, purification via centrifugation through a sucrosedensity gradient is utilized. The sucrose gradient may range from about0% sucrose to about 60% sucrose, preferably from about 5% sucrose toabout 30% sucrose. The buffer in which the sucrose gradient is made canbe any aqueous buffer suitable for storage of the fraction containingthe complexes and preferably, a buffer suitable for administration ofthe complex to cells and tissues. A preferred buffer is pH 7.0-8.0Hepes.

It is understood that in the present invention, preferred nucleic acidsequences are those capable of directing protein expression. Suchsequences may be inserted by routine methodology into plasmid expressionvectors known to those of skill in the art prior to mixing with cationicliposomes or liposomes and polycation to form the lipid-comprising drugdelivery complexes of the present invention. The amount of nucleic acidmixed together with cationic liposomes or with cationic liposomes andpolycation may range from about 0.01 μg to about 10 mg, preferably fromabout 0.1 μg to about 1.0 mg. It is understood that where the nucleicacid of interest is contained in plasmid expression vectors, the amountof nucleic acid recited above refers to the plasmid containing thenucleic acid of interest.

The purification of the nucleic acid/lipid and nucleicacid/lipid/polycation complexes of the present invention serves toconcentrate the nucleic acids and lipids contained in the resultantcomplexes from about 50-fold to about 500-fold such that the lipidcontent contained in the complexes may be as high as about 40 μmol/mland the nucleic acid content may be as high as about 2 mg/ml.

The nucleic acid/lipid/polycation complexes of the present inventionproduce particles of varying diameters upon formulation. As pointed outin the Background of Invention smaller particles tend to show greatersize stability than larger particles. Furthermore, smaller particles maybe more suitable for use as nucleic acid delivery vehicles. Particlediameters can be controlled by adjusting the nucleicacid/lipid/polycation ratio in the complex. FIG. 20 illustrates thisfact for Lipid:Protamine Sulfate:DNA complexes. The diameter of thecomplexes produced by the methods of the present invention is less thanabout 400 nm, preferably less than about 200 nm, and more preferablyless than 150 nm.

Nucleic acid/lipid/polycation ratios in the present invention furtheraffect the biological activity of the complexes. The in vitro and invivo transfection of mammalian cells can be facilitated by adjusting therelative amounts of nucleic acid, lipid and polycation. FIGS. 21, 22 and23 present data demonstrating this effect for the Lipid/ProtamineSulfate/DNA mediated transfection of HeLa and SKOV-3 cells. Where thepolycation is Protamine Sulfate and the nucleic acid is DNA, the nucleicacid/polycation ratio for the complex is between 1:0.01 and 1:100. Thenucleic acid/polycation ratio is preferably between 1:0.1 and 1:10, morepreferably between 1:0.5 and 1:5 and most preferably between 1:1 and1:3.

The complexes formed by the methods of the present invention are stablefor up to about one year when stored at 4° C. The complexes may bestored in 10% sucrose or a 5% dextrose solution upon collection from thesucrose gradient or they may be lyophilized and then reconstituted in anisotonic solution prior to use. In a preferred embodiment, the complexesare stored in solution. The stability of the complexes of the presentinvention is measured by specific assays to determine the physicalstability and biological activity of the complexes over time in storage.The physical stability of the complexes is measured by determining thediameter and charge of the complexes by methods known to those ofordinary skill in the art, including for example, electron microscopy,gel filtration chromatography or by means of quasi-elastic lightscattering using, for example, a Coulter N4SD particle size analyzer asdescribed in the Examples. The physical stability of the complex is“substantially unchanged” over storage when the diameter of the storedcomplexes is not increased by more than 100%, preferably by not morethan 50%, and most preferably by not more than 30%, over the diameter ofthe complexes as determined at the time the complexes were purified.

Assays utilized in determining the biological activity of the complexesvary depending on what drug is contained in the complexes. For example,if the drug is nucleic acid encoding a gene product, the biologicalactivity can be determined by treating cells in vitro under transfectionconditions utilized by those of ordinary skill in the art for thetransfection of cells with admixtures of DNA and cationic liposomes.Cells which may be transfected by the complexes includes those cellswhich may be transfected by admixture DNA/liposome complexes. Theactivity of the stored complexes is then compared to the transfectionactivity of complexes prepared by admixture. If the drug is a protein,then activity may be determined by a bioassay suitable for that protein.

It is further understood by those of skill in the art that the complexesof the present invention may be used in vivo as vectors in gene therapy.

Therapeutic formulations using the complexes of the invention preferablycomprise the complexes in a physiologically compatible buffer such as,for example, phosphate buffered saline, isotonic saline or low ionicstrength buffer such as 10% sucrose in H₂O (pH 7.4-7.6) or in Hepes (pH7-8, a more preferred pH being 7.4-7.6). The complexes may beadministered as aerosols or as liquid solutions for intratumoral,intravenous, intratracheal, intraperitoneal, and intramuscularadministration.

Any articles or patent referenced herein are incorporated by reference.The following examples illustrate various aspects of the invention butare intended in no way to limit the scope thereof.

EXAMPLES

Materials

DOPE was purchased from Avanti Polar Lipid, Inc. (Alabaster, Ala.).pRSVL, a plasmid which encodes the luciferase gene under the control ofRous sarcoma virus long terminal repeat, (De Wet, J. R. et al. (1987)Mol. Cell. Biol., 7: 725-737) was amplified in E. coli and purifiedusing the standard CsCl-EtBr ultracentrifugation method (Sambrook, J.,Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual(2d ed) Cold Spring Harbor Laboratory Press: New York (1989)). All thetissue culture media were obtained from Gibco BRL (Gaithersburg, Md.).Human embryonic kidney 293 cells, CHO (Chinese Hamster Ovary cells), BL6and BHK (Baby Hamster Kidney cells) cells were from American TypeCulture Collection (Rockville, Md.). Mouse lung cells (MLC) are primaryculture cells originally derived from the lung of a Balb/c mouse by Dr.S. Kennel (Oak Ridge National Laboratory, TN). 293, BL6, BHK and MLCcells were cultured with DMEM media, CHO cells were cultured with F12media, and C3 Hela cells were cultured in RPMI-1640 medium. All mediawas supplemented with 10% fetal bovine serum (Hyclone Laboratories,Inc., Logan, Utah) and 100 unit/ml penicillin and 100 μg/mlstreptomycin. Poly-L-lysine hydrobromide (MW 3000 and MW 25,600) andother chemicals were from Sigma (St Louis, Mich.).

DC-Chol was synthesized according to the method of Gao and Huang (1991)(Gao, X., and Huang, L. (1991) Biochem. Biophys. Res. Commun., 179:280-285) with modifications in the purification steps as follows: afterthe reaction, 10 ml hexane was added and the mixture was extracted threetimes with 10 ml water. The organic phase was collected and dried undervacuum at 4° C. The resulting solid was dissolved in a minimal amount ofabsolute ethanol with heat, and recrystallized in acetonitrile at 0/C.The purity of DC-Chol was at least >95%, as analyzed by TLC method and¹H-NMR and the yield of about 70% was a significant improvement overthat of the previously reported method of Gao, X., and Huang, L. ((1991)Biochem. Biophys. Res. Commun., 179: 280-285).

Methods

Preparation and Purification of Complexes

Cationic liposomes at a 20 mM total lipid concentration were preparedfrom DC-Chol and DOPE at various ratios by a sonication method,according to a published procedure (Gao, X., and Huang, L. (1991)Biochem. Biophys. Res. Commun., 179: 280-285). Trace amount of ³Hcholesteryl hexadecyl ether (Amersham, Arlington Heights, Ill.) wasincluded for quantitation purpose. The size of these liposomes wasbetween 100 to 150 nm in diameter, as determined by quasi-elastic lightscattering using a Coulter N4SD particle sizer (Coulter Electronics,Inc., Hialeah, Fla.). Unless indicated otherwise in the followingExamples, DNA/lipid complexes were prepared at a typical laboratoryscale by adding amounts of free DC-Chol/DOPE liposomes as indicated ineach Example in a volume of 1 ml of a 2 mM Hepes buffer (pH 7.6) to a15×7.5 polystyrene culture tube (Baxter, McGraw Pare, Ill.), amicro-magnetic stirrer was placed in the tube, and the solution was wellmixed. Amounts of pRSVL DNA as indicated in each Example were then addeddropwise from a stock solution (0.2 mg/ml, in 2 mM Hepes buffer, pH 7.6)to the liposome solution over a period of 3 min. Trace amounts of pRSVLlabeled with ³²P using a nick translation kit (Promega, Madison, Wis.)and ³²P dCTP (Amersham, Arlington Heights, Ill.) was included for thepurpose of quantitation.

To prepare purified lipid/PLL/DNA complexes, an amount of the above 0.2mg/ml DNA solution as indicated in each Example was added to 1 mlPLL/liposome mixture containing amounts of liposomes and PLL asindicated in each Example. DNA/lipid complexes were loaded on the top ofa sucrose step gradient composed of 0.5 ml each of 5%, 7.5%, 10% and 15%sucrose (w/w) and DNA/lipid/PLL complexes were loaded on top of asucrose step gradient composed of 0.5 ml each of 5%, 10%, 15%, 20%, 25%and 30% sucrose (w/w). The DNA/lipid and DNA/lipid/PLL complexes werethen purified from free lipid and PLL by ultracentrifugation at 100,000g for 30 min at 4° C. After centrifugation, fractions of 200 μl weretaken from the top to the bottom of the tube. Aliquots from eachfraction were assayed for both ³H and ³²P radioactivity using ascintillation counter. Fractions that contained peak value of the ³²Pwere collected and pooled. These pooled fractions were then assayed forparticle size and for transfection activity.

In Vitro Transfection Assay

The biological activity of the above complexes were assayed by in vitrotransfection of cells in the Examples as follows. Briefly, cells grownin 48 well plate were incubated with DNA/lipid complex diluted in 0.5 mlCHO—S—SFM (Gibco BRL) or with admixture DNA/liposome complex preparedaccording to Gao and Huang (1991) (Gao, X., and Huang, L. (1991)Biochem. Biophys. Res. Commun., 179: 280-285). For transfection of pRSVLDNA using DC-Chol liposomes in the presence of PLL, liposomes were firstmixed with PLL, then complexed with DNA. All transfections wereperformed for 4 hours at 37° C. After transfection, cells were furthercultured for 36 hours in the appropriate media containing 10% fetalbovine serum. Cells were then washed with PBS and lysed with 100 μl of1× lysis buffer provided by a luciferase assay kit (Promega, Madison,Wis.). A 4 μl sample of the lysate was assayed for luciferase activityusing 100 μl substrate solution from the reconstituted luciferase assaykit and an AutoLumat LB953 luminometer (Berthold, Germany). Proteinconcentration from each lysate was assayed by a Coomassie blue dyemethod according to the manufacturer's protocol (Pierce, Rockford,Ill.).

Example 1

The Size of the DNA/Lipid Complex is Determined by the Ratio of DNA toLipid

This experiment was conducted to show that the size of the DNA-lipidcomplex formed by admixture changed as the ratio of DNA mixed withliposome varied. In brief, pRSVL plasmid DNA (2 μg) was mixed withvarying amounts of DC-Chol/DOPE (3:2) liposomes in 2 mM Hepes buffer atpH 7.6 in a final volume of 500 μL and after 7 minutes, the size of thecomplex was determined with a Coulter N4SD laser light scatteringparticle sizer operating in the unimodel mode.

As shown in FIG. 1, large aggregates did not form when DNA was in excess(ratios of liposome to DNA less than 7) but at ratios of lipid to DNAthat were charge neutral (˜10), the size of the complex reached amaximum. In addition, when the ratio of liposome-to-DNA was keptconstant at 10 nmoles/μg, the size of the complex increased as both DNAand liposome concentration increased and eventually formed precipitates.However, when the ratio of liposomes to DNA was increased, the size ofthe complex was progressively reduced until the size of the complexbecame constant (250-300 nm) when the ratio of liposome-to-DNA exceeded25 nmoles lipid/μg DNA. This result may be due to the fact that the DNAwas perhaps coated by excess liposomes and therefore aggregation betweenthe complexes did not occur.

Based on the data presented in FIG. 1, lipid/DNA complexes were preparedusing a liposome-to-DNA ratio of 50 nmoles/μg by slowly adding a DNAsolution of 200 μg/ml to an excess amount (10 μmols) of liposome. Thesize of the complex formed was about 250 nm. When the ratio ofliposome-to-DNA was changed to 25 nmoles/m, the size of the complexincreased to about 350 nm. The complexes formed using either the 25nmole/μg or 50 nmole/μg ratios appeared to be physically stable since noprecipitates formed during storage for four weeks at 4° C.

Example 2

Purification of DNA/Lipid Complexes

When DNA was mixed with liposomes at a ratio of 1 μg/50 nmoles, anexcess of free liposomal lipids was observed to co-exist with theDNA/lipid complex. Since excess free lipids are toxic to cells, anexperiment was conducted to determine if free liposomal lipids could beseparated from the DNA/lipid complex by a density gradientultracentrifugation method. In brief, free liposomes (10 μmoles ofDC-Chol/DOPE (2:3) in a volume of 2 ml); free DNA (50 μg pRSVL in avolume of 2 ml) and DNA/lipid complex formed by mixing 20 μl molesDC-Chol/DOPE (2:3) and 0.4 mg pRSVL plasmid DNA (50 nmoles/μg) were eachcentrifuged for 30 minutes at 4° C. at 100,000 g over a gradientconsisting of 0.5 ml each of 5%, 7.5%, 10% and 15% sucrose (W/W).Fractions of 200 μl were then collected from the top to the bottom ofthe tube and assayed for the distribution of DNA marker (³²P, ▪) andlipid marker (3H, ◯). FIGS. 2A and 2B show the results of typicalseparations of free liposomal lipid, free DNA (FIG. 2A) and DNA/lipidcomplex (FIG. 2B) on the sucrose gradient. The results presented in FIG.2B show that after centrifugation, the complex formed a major band atthe 10% sucrose layer. By comparison, FIG. 2A shows that most of theradioactivity of the free DNA or free liposomal lipids distributed atthe top half of the tube and did not enter the sucrose gradient. Inaddition, although the peak of ³H and the peak of ³²P in FIG. 2Bco-existed at fraction number 16, there was a significant amount of the³H distributed in fractions 1 to 10 indicating that the excess freeliposomal lipids were well separated from the DNA/lipid complex.

Example 3

Physical Stability of Purified Lipid/DNA and Lipid/PLL/DNA Complexes

DNA/lipid complexes were formed by mixing 20 μl moles of liposomes ofvarious DC-Chol/DOPE compositions (see Table 1) with 0.4 mg pRSVLplasmid DNA at a ratio of 1 μg DNA/50 nmoles lipid. Lipid/PLL/DNAcomplexes were formed by mixing 4 μmoles of liposomes of variousDC-Chol/DOPE compositions with 1 mg PLL (MW=3000) and 0.4 mg pRSVLplasmid DNA. Both complexes were then purified from free lipids, freeDNA and free PLL by sucrose gradient centrifugation as described in theMethods section. The peak fractions were collected and pooled. Pooledsamples were then assayed for diameter immediately after collection (0days) or after storage in 10% sucrose at 4° C. for 120 days. Table 1shows the results of these assays.

TABLE 1 Physical stability of purified lipid/DNA and lipid/PLL/DNApre-complexes Liposome Size Purified composition PLL (nm) complex RatioRecovery (DC-chol/ (μg/ Day Day of lipid/DNA of DNA DOPE) μgDNA) 0 120(nmoles/μg) (% Total) 2:8 0 168 280 23.2 51 3:7 0 187 252 14.0 66 4:6 0175 195 12.7 73 5:5 0 174 210 13.2 70 6:4 0 198 232 10.1 69 2:8 2.5 165287 20.8 17 3:7 2.5 99 101 19.2 22 4:6 2.5 138 132 38.3 29 5:5 2.5 184178 22.4 27

The data presented in Table 1 shows that purified lipid/DNA andlipid/PLL/DNA complexes were small (under 200 nm) in size at day 0 andthat their size did not increase dramatically with storage. Further, theratios of DNA-to-lipid in the purified complexes was between 10 to 23nmoles lipid/μg DNA depending on the composition of the liposomes usedand this ratio did not change after storage for 120 days. A reciprocalrelationship between the concentration of DC-Chol in the liposomes andthe amount of the lipid in the complex was also observed indicating thatliposomes enriched with DC-Chol show stronger DNA binding or chargeneutralizing activity than liposomes less enriched with DC-Chol. Thefar-right hand columns shows recovery of ³²P-labeled DNA in theDNA/lipid and DNA/lipid/PLL complexes following their purification onthe sucrose density gradient. The results show that recovery of DNA inthe non-PLL containing complexes was higher than that observed for thePLL-containing complexes.

Example 4

Biological Activity of the Purified Complexes in Various Cells

Since the DNA/lipid complexes formed by a mixture of liposomes to DNAhaving a high lipid to DNA ratio were both small and stable over time,experiments were conducted to compare the transfection activity of thesecomplexes to the activity of DNA/liposome complex prepared by theadmixture method.

In one experiment, CHO cells cultured in 48 well-plates were treated for4 hours with an admixture of either 1 μg pRSVL and 10 nmolesDC-Chol/DOPE liposomes of different lipid compositions alone (◯) ortogether with 1 μg PLL (MW=3,000) (□) or, the cells were treated withpurified DNA/lipid complex (•) or purified DNA/lipid/PLL complex (▪)formed by mixing 1 μg DNA with 50 nmoles DC-Chol/DOPE liposomes(DNA/lipid complex) or with 10 nmoles DC-Chol/DOPE liposomes and 1 μgPLL (DNA/lipid/PLL complex) followed by centrifugation through a sucrosedensity gradient as described in the Methods section. 36 hours aftertreatment, cells were lysed in 100 μl lysis buffer and 4 μl of thelysate was assayed for luciferase activity using 100 μl of luciferasesubstrate solution. Luciferase activity was then counted over a periodof 20 seconds. The results presented in FIG. 3 show that the mostpreferred liposome composition for transfecting CHO cells was 40%DC-Chol and 60% DOPE. In addition, in the presence of additional 1 μgpoly-L-lysine (PLL, MW=3,000), a 2-7 fold enhancement of thetransfection activity was seen in most cases. Of particular interest,the activity of the purified DNA/lipid complex was similar to that ofthe admixture DNA/lipid complex when the same amount of DNA was added tocells. However, the transfection activity of the purified DNA/lipid/PLLcomplex was about 30% to 50% lower than that of the DNA/liposome/PLLcomplex prepared by the admixture procedure.

In order to determine that the results obtained in CHO cells were notcell-specific, the transfection activities of the purified DNA/lipid andDNA/lipid/PLL complexes in two other cells, BHK and mouse lung cells(MLC), were compared to that of DNA/liposome complexes formed byadmixture.

In brief, cells (either BHK or MLC) grown in 48-well plates at 60%confluency were transfected with 1 μg pRSVL complexed with 10 nmoles ofDC-Chol liposomes (admixture complex), with the same amount of DNA mixedwith liposomes at a DNA/liposome ratio of 1 μg/50 nmols to producepurified DNA/lipid complex or with purified DNA/lipid/PLL complexprepared at a DNA/liposome/PLL ratio of 1 μg/10 nmols/2 μg. Cells werethen harvested at 36 hours post-transfection, and the luciferaseactivity of the transfected cell lysates was determined as described inthe Methods section. The results of these experiments are shown inTables 2 and 3.

TABLE 2 Expression of luciferase gene in BHK cells transfected withpRSVL Luciferase Activity (Relative Light Units × 10⁻³) LiposomeAdmixture Purified Purified composition DNA/liposome DNA/lipidDNA/lipid/PLL (DC-Chol/DOPE) complex complex complex 2:8 91.8 ± 9.5110.1 ± 5.2 214.6 ± 41.1 3:7  61.2 ± 19.9  1886.8 ± 266.7 151.7 ± 62.94:6 438.2 ± 14.4 1638.8 ± 63.9 446.3 ± 16.9 5:5 837.8 ± 8   1015.0 ±41.2 234.2 ± 46.4

TABLE 3 Expression of luciferase gene in mouse lung cells transfectedwith pRSVL Luciferase Activity (Relative Light Units × 10⁻³) LiposomeAdmixture Purified Purified composition DNA/liposome DNA/lipidDNA/lipid/PLL (DC-Chol/DOPE) complex complex complex 2:8 1.1 ± 0.7 0.4 ±0.2  0.3 ± 0.1 3:7 1.5 ± 1.0 0.3 ± 0.0  4.1 ± 1.3 4:6 3.1 ± 0.2 2.0 ±0.3 14.6 ± 3.1 5:5 0.1 ± 0.0 1.5 ± 1.2 10.1 ± 2.3

Interestingly, for the BHK cell line, the transfection activity of thepurified DNA/lipid complex was significantly higher than that of theDNA/liposome complex formed by admixture. For cells such as MLC, whichare difficult to transfect, purified complexes made fromDNA/liposome/PLL mixtures were apparently superior to admixturecomplexes and to purified DNA/lipid complexes made without PLL.

In order to determine whether lipid/PLL/DNA complexes could be madeusing different ratios of lipid and nucleic acid and a differentmolecular weight PLL than that used in the previous examples, thefollowing experiment was conducted. Lipid/poly-L-lysine/DNA complex wasprepared from 20 μg pRSVL plasmid DNA, 10 μg poly-L-lysine (MW 25,600),and DC-chol/DOPE liposomes (4.5/5.5 molar ratio) at the ratios of lipidto DNA shown in Table 4. The resulting complexes were then purified bysucrose gradient ultracentrifugation as described in the methodssection. An aliquot of the purified complex containing 0.5 μg of DNA wasused to transfect CHO cells, and luciferase activity was then measured.The results of this experiment are shown below in Table 4.

TABLE 4 Effect of lipid/DNA ratio on purified complex containingpoly-L-lysine (MW 25,600) Composition of Transfection Ratio purifiedcomplex Size of activity^(b) (nmoles lipid/ (nmoles lipid/ purified(counts (SD) × μg DNA) μg DNA) complex (nm) 10⁻³) 3.3 1.1 89 108 (5) 6.6 2.5 98 6,065 (604) 12.5 4.3 101 5,846 (668) 20.0 9.6 35 7,633 (977)

The results show that in the presence of increased amounts ofpolycation, lower ratios of lipid to DNA may be used to produceDNA/lipid/polycation complexes having appreciable transfection activity.

Example 5

Transfection Activity of Stored Complexes

CHO cells cultured in 48 well-plates were treated for 4 hours withadmixture of 1 μg pRSVL and 10 nmoles of DC-Chol/DOPE liposomes ofdifferent DC-Chol/DOPE compositions alone (◯) or together with 1 μg PLL(mw=3,000) (□), or with purified DNA/lipid (•) or DNA/lipid/PLL (▪)complexes stored at 4° C. for 130 days in 10% sucrose. The purifiedcomplexes had been formed by mixing 1 μg pRSVL and 50 nmoles ofDC-Chol/DOPE liposomes of different DC-Chol/DOPE compositions alone(DNA/lipid) or with 10 nmole of DC-Chol/DOPE liposomes and 1 μg PLL(DNA/lipid/PLL complex) followed by centrifugation through a sucrosedensity gradient as described in the Methods section. The resultspresented in FIG. 4 show that the luciferase activity of cell lysatesprepared from cells transfected with the stored DNA/lipid andDNA/lipid/PLL complexes was comparable with the luciferase activityobserved in cell lysates of cells transfected with the correspondingcomplexes prepared by admixture.

Example 6

Comparative Cytotoxicity of DNA/Liposome Complexes Prepared by Admixtureto that of Purified DNA/Lipid Complexes

Cell toxicity of the different complexes was studied in CHO cells asfollows. CHO cells were treated with admixture DNA/liposome complex (◯),admixture/liposome/PLL complex (□); purified DNA/lipid complex (•); orpurified DNA/lipid/PLL complex (▪). The admixture complexes were formedby mixing 1 μg pRSVL DNA with 10 nmoles DC-Chol/DOPE liposomes ofdifferent DC-Chol/DOPE compositions alone or together with 1 μg PLL(mw=3,000). The purified complexes were formed by mixing 1 μg pRSVL DNAwith 50 nmoles DC-Chol/DOPE liposomes alone (DNA/lipid complex) or with10 nmoles DC-Chol/DOPE liposomes and 1 μg PLL (DNA/lipid/PLL complex)followed by centrifugation through a sucrose density gradient asdescribed in the Methods section. 36 hours after treatment, the cellswere lysed, protein was extracted and then quantitated by a Coomassieblue dye method.

FIG. 5 shows the results of this experiment where the amount of totalextractable protein recovered at the end of the experiment serves as anindicator of the portion of the cells which survived after the indicatedtreatment. The data presented shows that while the purified complexappeared to be slightly more toxic to the cells than the admixtureDNA/liposome complex, morphologically, the transfection did not causeany serious cytotoxic effects in cells treated with either admixturecomplexes or the purified complexes, except that the cells treated withpurified complexes containing high mole % DC-Chol were less confluent atthe end of the experiment.

Example 7

In Vivo Transfection of Tumors by Purified DNA/Lipid Complexes

3×10⁶ human ovarian carcinoma cells were injected subcutaneously intoSCID mice at day 0.14 days later, 100 μl solutions containing pUCCMVCATDNA (30 μg) complexed with DC-Chol (3:2 DC-Chol:DOPE) liposomes (30nmoles) in the form of admixture (lanes 1 and 2) or the same amount ofDNA in the form of purified DNA/lipid complex (prepared from DNA andDC-Chol liposomes at ratios of 1 μg DNA/25 nmoles lipid) were directlyinjected into tumors. Animals were sacrificed 2 days later and tumorextracts containing 100 μg protein were assayed for CAT activity at 37°C. according to Ausubel, et al. (1991) Current Protocols in MolecularBiology (Wiley, Boston), Vol. 1, pp. 9.6.2-9.6.5). The results show thatpurified complex, while prepared under non-optimal conditions, exhibitedin vivo transfection activity.

Example 8

Comparative Transfection Activity of Purified and UnpurifiedDNA/Lipid/PLL Complexes with Admixture DNA/Lipid Complex

The transfection activities of purified and unpurified DNA/Lipid/PLLcomplexes and admixture DNA/lipid complex in three cell lines (293, BL6and C3) were measured as follows:

Purified DNA/lipid/PLL complex was formed by mixing 1 μg pRSVL DNA with10 nmoles DC-Chol/DOPE liposomes (2:3 mol/mol) and 1 μg PLL (MW=25,600)followed by purification via centrifugation through a sucrose densitygradient as described in the Methods section.

Unpurified DNA/lipid/PLL complexes were formed by mixing 100 μg pRSVLDNA with 80 μg PLL (MW=25,600) and 1702 nmol DC-Chol/DOPE (2:3 mol/mol)liposomes (i.e. a DNA/lipid/PLK ratio of 1 μg DNA/17 nmol lipid/0.8 μgPLL) in a final volume of 500 μl of water. 20 μl of the unpurifiedDNA/lipid/PLL complex (i.e. 4 μg DNA, 3.2 μg PLL and 68.1 nmol lipid)was then added to 780 μl of serum free medium appropriate for the cellline to be transfected.

Admixture DNA/lipid complex was formed by mixing 1 μg pRSVL DNA with 10nmols of DC-Chol/DOPE (3:2 mol/mol) liposomes.

293, C3 and BL6 cells grown to 80% confluence in 24-well plates weretransfected for four hours at 37° C. with 1 μg of DNA in the form ofpurified DNA/lipid/PLL complex, admixture DNA/lipid complex orunpurified DNA/lipid/PLL complex. After transfection, cells were furthercultured for 36-48 hours in the appropriate media containing 10% fetalcalf serum. Luciferase activity was then measured as described in theMethods section.

The results presented in FIGS. 7A (293 cells) 7B (C3 cells) and 7C (BL6cells) demonstrate that the unpurified DNA/lipid/PLL complex exhibitedthe highest transfection activity in all three cell lines tested.

Materials and Methods for Examples 9-14 and in FIGS. 8-19

Plasmid DNA

The plasmid DNA used for all experiments consisted of a luciferase (LUC)reporter gene driven by the human cytomegalovirus (CMV) immediate earlypromoter and cloned into a pUK21 backbone. Plasmid DNA was prepared bygrowing bacterial stocks in a glycerol enriched Terrific Broth (TB)media. The bacteria was subject to detergent and alkaline lysis,propanol precipitation and treatment by high salt to remove highmolecular weight RNA. The resulting supernatant was subjected to sizeexclusion chromatography to separate low molecular RNA from plasmid DNA.The DNA was shown to be absent of chromosomal DNA or RNA by gelelectrophoresis and had an A_(260/280) ratio between 1.80-1.90 asdetermined spectrophotometrically.

Liposomes

DC-Chol was synthesized as previously described (Gao and Huang, (1991)Biochem. Biophys. Res. Commun., 179:280-285)). DOPE was purchased fromAvanti Polar Lipids, Inc. DC-Chol liposomes were produced bymicrofluidization at a 6:4 molar ratio (DC-Chol to DOPE) and to aconcentration of 2 μmol/ml (1.2 mg/ml of total lipid) as previouslydescribed in Int. J. Pharm. 14:34:30 1996.

Polycations

Poly-L-Lysine (MW 18,000-19,200), Protamine Free Base (Grade IV),Protamine Phosphate (Grade X), Protamine Chloride (Grade V), ProtamineSulfate (Grade II), Protamine Sulfate (Grade III), and Protamine Sulfate(Grade X) were all obtained from Sigma Chemical Co. Protamine Sulfate,USP was obtained from Elkins-Sinn, Fujisawa or from Eli Lilly andCompany. All of the protamines were from salmon sperm except the gradeIII which was from herring.

Transfection of Mammalian Cells In Vitro

CHO cells were seeded into a 48-well plate and grown in F-12 mediasupplemented with 10% Fetal Bovine Serum (FBS) in a 37/C incubator in a5% CO₂ atmosphere. The cells were exposed to DNA complexed to DC-Cholliposomes with or without a polycation such as protamine or poly-lysineand allowed to transfect for 5.5-6 hours after which the transfectionmedia (Hanks buffer) was replaced with fresh F-12 media supplementedwith 10% Fetal Bovine Serum (FBS). Typically, 8 μg of Protamine (or 4 μgof poly-lysine) were mixed with 4 μg of DNA in a 1 ml volume of Hanksbuffer in a 4 ml tube for approximately 5-15 run at room temperature. Asecond 4 ml tube containing 30 nmol of DC-Chol liposomes in 1 ml ofHanks buffer was added to the 1 ml sample of DNA/polycation and allowedto complex for 5-15 minutes at room temperature. After mixing, 0.5 mlaliquots (containing 1 μg DNA, 2 μg protamine or 1 μg poly-1-lysine, and7.5 nmol of DC-Chol liposomes) were added to each well of the 48-wellplate. The cells were allowed to incubate for a total of 35.5-38.5 hrsprior to being assayed for luciferase activity. Each point shown inFIGS. 8 and 9 represents the mean (with standard deviation) luciferaseactivity of three to four data points and are normalized to proteincontext.

In vitro transfection assays for HeLa cells (available from ATCC) andthe SKOV-3 human ovarian carcinoma cell line (available from ATCC) wererun under the same conditions as described above for CHO cells.

Luciferase Assay

CHO cells were allowed to transfect for 4-8 hours in transfection mediaand then allowed to incubate in F12+10% FBS for an additional 32-44hours. The media was then aspirated and the cells were washed once witha 0.9% sodium chloride solution. The cells were lysed with 100 μl oflysis buffer (2 mM EDTA, 100 mM Tris, 0.05% Triton X-100) per well andsubjected to one cycle of freeze-thaw. The cell lysates were collected,briefly centrifuged to pellet the cellular debris, and 10 μl was usedfor the luciferase assay. The samples were loaded into an AutoLumatLB953 (Berthold) Luminometer, 100 μl of the luciferase substrate(Promega) was added to the cell lysate, and the relative light units(RLU) of each sample were counted for 20 seconds. Samples were alsoassayed by Coomassie Blue Plus Protein Reagent (Pierce) and normalizedto the protein content in each sample.

Example 9

Comparison of the Ability of Protamine Sulfate USP (Elkins-Sinn) andPoly-L-Lysine to Increase Transfection Activity in CHO Cells

Protamines are naturally occurring cationic proteins which arecharacterized as being small (mw=4,000), extremely basic, and containinga large amount of arginine (Ando et al., (1973) in Protamines—Isolation,Characterization, Structure and Function (Springer-Verlay, New York) pp.3-87)). Protamines are only found in the head of mature sperm and theirsole function is to condense DNA such that it can be efficientlypackaged and ultimately expressed within the nucleus of an egg.Protamines are typically purified from fish (salmon or herring) sperm.These proteins are approximately 30-35 amino acids in length and abouttwo thirds (⅔) of these residues are arginine. The arginine residues arefound in clusters of 4-6 in four distinct regions. About one fourth ofthe remaining residues are hydrophobic, resulting in evenly spacedhydrophobic and hydrophilic regions. The result of this uniquearrangement of amino acids is that the protamine molecule is forced toassume an α-helical structure in the presence of nucleic acids. Thespacing of the amino acids in protamine parallels the spacing of thebases in DNA. As a result, the protamine α-helical structure alignsalong the major groove of DNA and associates strongly with the DNAwithout changing its conformational state. It can also cross-link withadjacent DNA strands producing a highly ordered, condensed DNA/protaminecomplex (Warrant and Kim, (1978) supra). While poly-lysine andpoly-arginine have also been shown to condense DNA, they lack the finestructural characteristics found in protamine. As a result, thecondensation by poly-lysine or poly-arginine results in a complex whichis structurally different. This results in a change in theconformational and crystalline state of DNA from the biologically activeB-form (as found in protamine) to an inactive crystalline A-form(Subirana, (1983) in the Sperm Cell ed. Andre, J. (Martainui, Nizeroff,the Hague), pp. 197-213)).

Accordingly, an experiment was conducted in which increasing amounts ofsalmon sperm protamine sulfate, USP or poly-L-Lysine were added to 1 μgof DNA and allowed to associate prior to the addition of 7.5 nmol ofDC-Chol liposomes.

As shown in FIGS. 8A and 8B, increasing amounts of poly-L-Lysineresulted in an increase in transfection activity reaching a constantlevel of activity at 1 μg of poly-L-lysine per 1 μg of DNA.

Increasing amounts of Protamine Sulfate, USP also resulted in anincrease in expression, reaching a constant level of expression at 2 μgof Protamine Sulfate, USP per 1 μg of DNA (see FIGS. 8A and 8B).However, in sharp contrast to the previously published report of Gao andHuang ((1996) Biochem., 35:1027-1036), in which transfection activitywith protamine free base was consistently lower than that observed withpoly-L-Lysine, the level of expression achieved with Protamine Sulfate,USP/DNA/liposome complexes was unexpectedly 4-5 fold higher than thelevels seen with poly-L-lysine and 40-fold higher than that observed forDNA/liposome complexes without polycation.

Example 10

Effect of Different Types of Protamine on Transfection Activity

In order to further explore this unexpected difference in the ability ofprotamine free base and protamine sulfate USP to potentate transfectionactivity, the ability of various types of protamine to potentiate geneexpression in CHO cells was compared to standard DNA/liposome complexesas well as complexes supplemented with poly-L-Lysine.

As shown in FIG. 9, the potentiation of gene expression by differenttypes of protamine varied considerably with protamine phosphate and freebase showing minimal if any increase in gene expression over that seenin the absence of any polycation while protamine sulfates showed a 3-5fold increase in gene expression over poly-L-lysine and increased geneexpression by 60-fold over background (DNA/lipid complex without apolycation (poly-L-lysine or protamine sulfate)). In addition, protaminechloride also showed an increase in gene expression over poly-L-lysine,albeit not as great an increase as that observed for protamine sulfates

Example 11

Amino Acid Analysis of Various Types of Protamine

In an effort to explain the large variation in transfection activity ofthe various protamines described above, the relative percentages ofamino acid residues (i.e., amino acid composition) present in protaminephosphate, chloride, free base, sulfate (grade III), and sulfate USP(Lilly and Elkins-Sinn) were determined for each compound. The resultsof this analysis are presented in Table 5. Predicted values arecalculated from a published sequence of salmon sperm (Warrant and Kim,(1978) Nature, 271:130-135).

TABLE 5 Amino Acid Composition of Various Salts of Protamine (expressedas percentage of composition) Sulfate, Sulfate Sulfate, USP Pre- FreePhos- Chlo- (Grade USP (Elkins- dicted Amino Acid Base phate ride III)(Lilly) Sinn) * Aspartate 0.23 0.55 0 0.12 0.50 0.03 0 Threonine 0.100.35 0 0 0 0 0 Serine 5.95 6.60 8.91 8.46 8.95 8.23 12.5 Glutamate 0.070.01 0 0 0.09 0 0 Glycine 6.69 7.07 7.04 6.56 7.30 6.36 6.25 Alanine1.44 1.76 1.49 0.94 1.73 1.32 0 Valine 4.69 4.57 4.61 4.93 4.43 4.316.25 Methionine 0.56 0.77 0.70 0.64 0.75 0.64 0 Isoleucine 1.26 1.231.33 0.81 1.31 1.20 0 Leucine 0.17 0.20 0 0 0.22 0 0 Histidine 0.10 0.110 0 0 0 0 Lysine 8.14 8.84 1.49 0.47 0.21 0.23 0 Arginine 61.82 59.6170.37 68.25 65.90 69.27 65.63 Proline 8.79 8.35 8.60 8.81 8.27 8.42 9.38

As can be seen, the amino acid composition of protamine free base isvery similar to that of the protamine phosphate. Strong similarity inamino acid composition is also observed among the three differentprotamine sulfates and protamine chloride. Of particular interest arethe differences in amino acid composition between the protamine sulfatesand protamine chloride and the protamine free base and phosphate. Themost striking difference is a 6-32 fold increase in lysine content inthe phosphate and free base form of protamine relative to the sulfatesand the chloride. An additional difference is a 1.5 fold increase in theamount of serine in the chloride and the sulfates relative to thephosphate and free base. Also observed is a decrease in the percentageof arginine present in the free base and phosphate in comparison to thesulfates and chloride. Of particular interest, the observed differencesin lysine content correlate with the variations in activity observed inFIG. 9.

Of clinical interest, protamines are naturally occurring compounds whichelicit rare, if any immune responses in the host and have been usedclinically for several decades in insulin delivery systems whereas allcurrent delivery systems utilize synthetic polymers having unknownsafety profiles in humans.

Example 12

In Vivo Transfection by DNA/Protamine Sulfate/Lipid Complexes

Use of PS/DNA/liposome formulations in vivo has been demonstrated byadministering various formulations intravenously to mice and assayingfor reporter gene based on luciferase activity. A formulation containingPS/DNA/DOTAP (0.8 μg/1 μg/23.27 μg) in 5% dextrose has been shown tohave a greater transfection activity than either PS/DNA (0.8 μg/1 μg)(data not shown) or complexes produced in the absence of PS (23.27 μgDOTA/μg DNA) (FIG. 10). Gene expression was highest in lung tissuecompared to kidney, spleen, liver, and heart. In vivo luciferaseactivity was positively correlated with increasing concentrations ofDOTAP and was maximal at a level of 11:1 (DOTAP/DNA mol/mol) (FIG. 11).Increasing the concentration of DNA also resulted in increased geneexpression but was associated with some toxicity above a dose of 75 μgof DNA and the death of one mouse at a dose of 100 μg of DNA (FIG. 12).In vivo gene expression appeared to be maximal at 6 hours followinginjection and subsequently declined thereafter (FIG. 13).

Example 13

In Vitro Optimization of Amounts of Protamine Sulfate, DNA, and DC-CholLiposomes for Maximal Transfection

Optimization of the protamine sulfate/DNA/DC-Chol liposome complexeswere examined in order to establish parameters for the complexformulation. As shown in FIGS. 8A and 8B, maximal activity was seen when2 μg of protamine sulfate was complexed with 1 μg of DNA and 7.5 nmol ofDC-Chol liposomes. In order to establish the optimal amount ofDNA/Protamine sulfate which can be delivered by a fixed amount ofDC-Chol liposomes, increasing amounts of DNA/PS (at a 1:2 w/w ratio)were complexed with 7.5 nmol of DC-Chol liposomes. As can be seen inFIG. 14, optimal transfection activity was achieved when 2 μg of DNA and4 μg of PS were complexed with 7.5 nmol of DC-Chol liposomes.

Having established that 2 μg of PS is required to optimally deliver 1 μgof DNA (FIGS. 8A and 8B), the amount of DC-Chol liposomes required toefficiently deliver this quantity of DNA/PS was determined. As shown inFIG. 15, using 1 μg of DNA complexed with 2 μg of PS, efficient deliverywas established with as little as 2.5 nmol of DC-Chol liposomes. Priorexperiments were performed at this DNA/PS ratio by using 7.5 nmol ofDC-Chol liposomes. Since it is thought that cellular toxicity is afunction of liposome concentration, it is preferential to use as minimala dose of lipids as needed to deliver a specified quantity of geneticmaterial. FIG. 16 suggests that as little as 2.5 nmol of DC-Cholliposomes is required to deliver a complex consisting of 1 μg of DNA and2 μg of PS. The results of FIG. 15 suggest that quantities of 2 μg ofDNA and 4 μg of PS can be delivered efficiently. This finding suggeststhat 2 μg of DNA complexed with 4 μg of PS would best be delivered by 5nmol of DC-Chol liposomes.

Example 14

The Ability of Protamine Sulfate to Increase the Transfection Activityof Several Different Cationic Liposome Formulations In Vitro

Having established the ability of PS to augment the transfectionactivity of DC-Chol liposomes (FIGS. 8A and 8B), several othercommercially available liposome formulations were also tested. FIG. 16shows the ability of Clonfectin (Clontech) to transfect CHO cells with 1μg of pUK 21 CMV LUC (□) and with a complex consisting of 2 μg of PS and1 μg of DNA (▪). The addition of PS results in a 5-20 fold increase inluciferase activity over the concentrations tested. FIG. 17 shows theability of DC-Chol liposomes to transfect CHO cells with 1 μg of DNA (□)and with a complex consisting of 2 μg of PS and 1 μg of DNA (▪). Theaddition of PS results in a 10-85 fold increase in luciferase activityover the concentrations tested. FIG. 18 shows the ability of Lipofectin(Gibco BRL) to transfect CHO cells with 1 μg of pUK 21 CMV LUC (□) and acomplex consisting of 2 μg of PS and 1 μg of DNA (▪). The addition of PSresults in a 15-105 fold increase in luciferase activity over theconcentrations tested. FIG. 19 shows the ability of DOTAP/DOPE (1:1mol/mol) liposomes to transfect CHO cells with 1 μg of pUK 21 CMV LUC(□) and with complex consisting of 2 μg of PS and 1 μg of DNA (▪). Theaddition of PS results in a 10-220 fold increase in luciferase activityover the concentrations tested.

While we have hereinbefore described a number of embodiments of thisinvention, it is apparent that the basic constructions can be altered toprovide other embodiments which utilize the methods and devices of thisinvention. Therefore, it will be appreciated that the scope of thisinvention is defined by the claims appended hereto rather than by thespecific embodiments which have been presented hereinbefore by way ofexample.

Example 15

Relationship Between Lipid:Protamine Sulfate:DNA Ratios and AverageParticle Size

A number of lipid:protamine sulfate:DNA formulations were made andanalyzed to establish the relationship between Lipid:ProtamineSulfate:DNA ratios and particle size. Complexes were formed between PSand pCMV-Luc plasmid DNA in incremental ratios varying from 0:0 to 4:0.Aliquots of DC-Chol HCl/DOPE (Aronex) suspended in 10% dextrose wereadded to the PS/DNA complex to produce Lipid:DNA ratios from 1:1 to10:1. The particle sizes of the resulting LPD complexes were taken atroom temperature on a Malvern Zetasizer when the complexes were firstformed (Day 0) and again after storage at 4° C. for 7 days. The datagenerated from this study are shown in FIG. 20.

The data from FIG. 20 indicate a correlation between Lipid:ProtamineSulfate:DNA ratios and particle size.

Particle sizes around 200 nm are preferable for coated pitinternalization.

Example 16

In Vitro Transfection Efficiency of Different LPD Formulations on HeLaand SKOV-3 Cells

HeLa and SKOV-3 cells were transfected with different LPD formulationsaccording to the protocol presented in the Materials and Methodssection. Transfection efficiency was measured using the Luciferase Assay48 hrs post transfection.

As depicted in FIG. 21, the addition of 2 μg Protamine Sulfate to a 1:1lipid-DNA complex increases luciferase activity in HeLa cells by 500fold. The addition of Protamine Sulfate to a 10:1 lipid-DNA complex toproduce a 10:2:1 LPD formulation increases luciferase by 50 fold. Noimprovement in transfection activity is observed beyond a ratio of 2:1Protamine:DNA.

FIG. 22 presents a comparative study of the transfection efficiency ofSKOV-3 cells using LPD and DC-Chol delivery vehicles. The n valuerepresents the number of individual experiments which were averaged forthis data table. LPD formulations produced consistently higherluciferase activity than did the DC-Chol formulations, with the 15:2:1LPD complex proving 8-10 fold more efficient that the best LD complex.

Example 17

Transfection of Mammalian Cells In Vivo

Nude mice between 6 and 12 weeks of age were inoculatedintraperitoneally with 2×10⁶ SKOV-3 human ovarian carcinoma cells in atotal injection volume of 0.5 mls of PBS. After 6-7 weeks of tumor cellengraftment animals were injected with different formulations ofpCMV-luc plasmid DNA and DNA/Lipid in a total volume of 1.0 ml (5%Dextrose final, isotonic solution). Animals were sacrificed 16 hourspost formulation injection. Tumor nodules were removed and lysed.Protein concentrations were determined according to the Luciferase Assaydescribed above.

FIG. 23 shows data comparing the in vivo transfection efficiency ofvehicle plus naked DNA to following LPD formulations: 0:0:1, 0:2:1,3:0:1, 3:2:1, 10:0:1, 10:2:1, 15:0:1 and 15:2:1. The RLU/mg valuesobtained for each individual mouse are denoted by circles, while theaverage RLU/mg values are shown as squares. Data show that the 15:2:1LPD complex produced the highest average expression level of theformulations tested, yielding a 3-4 fold higher expression value.

Example 18

LPD E1A Survivability Study

Nude mice were injected intraperitoneally with 2×10⁶ SKOV-3ip1 cells (ahuman ovarian carcinoma cell line selected as a subline from SKOV-3 dueto its more rapid growth and higher HER2/neu expression). At five dayspost implantation, a treatment regimen involving delivery of the E1Aexpression plasmid comprising the Ad5 E1A gene with its native promoter(see, e.g., Mien-Chie Hung et al., U.S. Pat. No. 5,651,964). Animalswere injected intraperitoneally on days 5, 6, and 7 with 1 ml of a 5%Dextrose vehicle comprising the formulations presented in FIG. 24: Group1 animals were repeatedly injected with the 5% Dextrose vehicle only;Group 2 animals were repeatedly injected with the equivalent amount ofDC-Chol/DOPE (6:4 ratio) and Protamine sulfate as one would find in thehigh dose 15:2:1 group but without the E1A expression plasmid present;Group 3 animals were repeatedly injected with 15 μg/dose of naked E1Aexpression plasmid, which was equivalent to the highest dose injected informulated vehicles; Group 4 animals were repeatedly injected with 15μg/dose of E1A expression plasmid compacted with Protamine sulfate at aratio of 2 μg Protamine/1 μg DNA, which was equivalent to the highestdose injected in the formulated vehicles; Group 14 animals wererepeatedly injected with a 1.5 μg/dose 15:2:1 LPD formulation; Group 15animals were repeatedly injected with a 5 μg/dose 15:2:1 LPDformulation; Group 16 animals were repeatedly injected with a 15 μg/doseLPD formulation. Animals were subsequently injected once a week with thesame formulation for the time listed on the X axis. Animals eithersurvived, died due to tumor burden, or were sacrificed prior to death ifdeath was imminent.

FIG. 24 shows that DNA must be formulated in a lipid vehicle to workproperly upon intraperitoneal injection (Groups 3 and 4). The datafurther demonstrate a positive correlation between the dose of E1A givenas a lipid formulation and the mouse survival rate (Groups 14, 15 and16).

1. A method for producing a noncovalent nucleic acid/lipid/polycationcomplex having a net positive charge at pH 6.0-8.0, the methodcomprising, providing a cationic lipid and a polycation to form a lipid/polycation complex, and providing a nucleic acid with thelipid/polycation complex in a ratio of nucleic acid to lipid topolycation which results in the production of the nucleicacid/lipid/polycation complex, wherein the polycation is a polycationicpeptide or a polycationic protein.
 2. The method of claim 1, wherein thecationic lipid is a cationic liposome.
 3. The method of claim 2, whereinthe cationic liposome further comprises a neutral lipid.
 4. The methodof claim 1, wherein the polycation has a molecular weight of 300 to200,000 Daltons.
 5. The method of claim 1, wherein the polycation is apolycationic peptide.
 6. The method of claim 4, wherein the polycationicpeptide is a synthetic polycationic peptide.
 7. The method of claim 5,wherein the polycationic peptide is a synthetic peptide derived fromSV40 large T antigen and has net positive charge and a nuclearlocalization sequence.
 8. The method of claim 5, wherein thepolycationic peptide comprises lysine residues.
 9. The method of claim5, wherein the polycationic peptide comprises polylysine.
 10. The methodof claim 1, wherein the polycation is a polycationic protein.
 11. Themethod of claim 10, wherein the polycationic protein is a histone. 12.The method of claim 1, wherein the polycation is a peptide comprising anuclear localization sequence of SV40 large T antigen.
 13. The method ofclaim 1, wherein the polycation comprises spermine or spermidine. 14.The method of claim 5, wherein the polycationic peptide comprises anatural polycationic peptide.
 15. The method of claim 10, wherein thepolycationic protein is a protamine.
 16. The method of claim 1, whereinthe ratio of lipid to nucleic acid ranges from about 0.1 nmol to 200nmol lipid per 1 μg nucleic acid.
 17. The method of claim 1, wherein theratio of lipid to nucleic acid ranges from about 1 nmol to about 25 nmollipid per 1 μg nucleic acid.
 18. The method of claim 1, wherein thepolycation:nucleic acid ratio is about 0.01 μg to about 100 μgpolycation to 1 μg nucleic acid.
 19. The method of claim 1, wherein thelipid/polycation complex is stored for up to 120 days prior to providingthe nucleic acid with the lipid/polycation complex.
 20. The method ofclaim 1, wherein the lipid/polycation complex is stored for up to a yearprior to providing the nucleic acid with the lipid/polycation complex.21. The method of claim 1, wherein the lipid/polycation complex isstored for between four weeks and a year prior to providing the nucleicacid with the lipid/polycation complex.
 22. The method of claim 1,wherein the polycation is up to about 500 amino acids in length.
 23. Themethod of claim 4, wherein the polycation is a peptide comprisingpolylysine.